FROM BULLETIN OF THE UNITED STATES BUREAU OF FISHERIES : VOLUME XXXII. 1912 
Dooumenl 784 ::::::::::::: Issued February 18. 1914. 



A NEW METHOD FOR THE DETERMINATION OF THE FOOD 

VALUE OF PROTEINS, WITH APPLICATION TO 

CYNOSCION REGALIS 

By George F. White and Adrian Thomas 



175 



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.46- 



^). OF D. 



A NEW METHOD FOR THE DETERMINATION OF THE FOOD 

VALUE OF PROTEINS, WITH APPLICATION 

TO CYNOSCION REGALIS. 

By GEORGE F. WHITE and ADRIAN THOMAS. 



It is frequently desired to compare the rate and course of digestion of various 
proteins by enzymes according to methods which, while not complicated, will give 
distinctive and reliable results. Van Slyke's" method for the determination of amino 
acids has been applied by White and Crozier* with evidently great success to a compari- 
son of the tryptic proteolysis of beef and several fish meats. The ease of manipulation 
/ of the apparatus, the brief time required for a determination, and the regularity of the 
experimental data, make the process generally useful. The conclusions drawn also 
conform with those from metabolism experiments, making the results of still greater 
value. 

The method which Sorensen'^ has proposed for the estimation of amino acids and 
polypeptides, by titration with caustic soda after the addition of formaldehyde, by its 
simplicity suggests itself for artificial digestion processes. It is the object of this article 
to show its application to the hydrolysis of Cynosdon rcgalis (squeteague, weakfish) 
bynrypsin, and to compare the results with those obtained by Van Slyke's method. 

The squeteague was boiled in water for a quarter of an hour, allowed to drain from 
excess of liquid, and preserved ice-cold. An analysis of two samples gave an average 
of 4.52 per cent nitrogen. 

The digestion was carried on in 250 c. c. volumetric flasks placed in a thermostat 
kept at temperature of 37.5° C. Enough meat to furnish i .5 g. of nitrogen was weighed 
out, ground up with water together with i g. of trypsin, and this mixture poured into 
the flask; 25 c. c. of N/ 10 sodium hydroxide solution was added, and the whole made up to 
250 c. c. with water. Trypsin is presumably most active in a medium made alkaline 
with sodium carbonate, but the presence of this salt would interfere with the titration 
for the amino acids where phenolphthalein must be used as an indicator, so that the 
alkalinity was insured by the presence of the hydrate. Separate mixtures were made 

o Van Slyke, D. D. : A method for quantitative determination of aliphatic amino groups. Journal of Biological Chemistry, 
v«.l. IX. p. 185-204. 1911. Baltimore. 

t White, G. F. &Cro2ier. W.; Comparative proteolysis experiments with trypsin. Journal of the American Chemical Sodety, 
vol. 33. p. 3042-2048. igii. Easton, Pa. 

«Sorensen, S. P. L.: Fermentstudien. BiochemischeZeitschrift. bd. 7. p. 45-101. 1907. Berlin. 

'77 



178 



BULLETIN OF THE BUREAU OF FISHERIES. 



and analyses run in duplicate for the time periods of i , K. 2, 5, and 8 hours, respectively. 
A sample similar to the above but containing no trypsin was also prepared and analyzed 
with the others according to the following method: At the end of the desired time of 
digestion, the mixtures were filtered and aliquot portions of the filtrate taken for the 
various tests; 10 c. c. were used for the determination of total soluble nitrogen by the 
Kjeldahl method; 10 c. c. for the amino nitrogen by Van Slyke's method; 20 c. c. were 
treated with 10 c. c. of 40 per cent formaldehyde solution and titrated to a distinct 
pink color with N/io sodium hydroxide solution. We found that in every case a distinct 
end point was obtained, duplicate analyses agreeing within 0.2 per cent. Finally, 
10 c. c. of the filtrate from the digested fish were completely hydrolyzed by prolonged 
digestion on the water bath with 40 c. c. of concentrated hydrochloric acid. This solution 
was evaporated to dryness, made up to 50 c. c. with water, 10 c. c. tested for amino 
nitrogen by Van Slyke's method, and 20 c. c. analyzed for amino acids by Sorensen's 
method. 

All the results as obtained above were corrected for amino nitrogen before and after 
complete hydrolysis, for total soluble nitrogen, and for amino acids as determined by 
titration with caustic soda, by carrying through the same experiments with trypsin and 
alkali, but with no protein. Correction was also made for the alkali required to 
neutralize the formaldehyde solution. 

In table i are presented the results obtained by applying Van Slyke's method to 
the tryptic proteolysis of squeteague. The average size of the peptides was calculated 
by dividing the amount of amino nitrogen present after complete hydrolysis with hydro- 
chloric acid by that in the solution before such hydrolysis. The last two columns of 
data show the increase with time of proportion of soluble to insoluble nitrogen and of 
amino to total soluble nitrogen, respectively. The average results of dupHcate analyses 
are given. 

Table I. — Total and Amino Nitrogen in Solutions op Cynoscion Regalis 
Hydrolyzed by Trytsin. 



Time in 
hours. 


Soluble 
nitrogen. 


Insoluble 
nitrogen. 


Amino 
nitrogen. 


Amino 
nitrogen 
after hy- 
drolysis. 


Average 

size of 

peptids. 


Soluble 


Amino 
looX 

soluble 
nitrogen. 


total 
nitrogen. 




I 

2 

s 
s 


0. 170 
1. 113 

1. 175 

I. "73 
I- j6i 
■■432 


1-330 
■385 
•340 
•327 
.139 
.06S 


0.017 
.230 
.250 
.2S9 
■3S7 
.400 


o.os6 
.464 
.464 
■464 


3-29 

2.02 
1.86 
1. 61 


11.32 
74-32 
78.34 
78.20 
90.72 
95-45 


ID. 00 
20.63 
21.27 
24.63 
26.23 
28.3s 


.646 


1-59 



In table 11 are given the average results of the analysis of the proteolyzed solutions 
according to S5rensen's method. In column 2 the figures represent cubic centimeters 
of N/io sodium hydroxide solution required for neutralization after addition of formal- 
dehyde solution. Column 3 is the same for the solutions after complete hydrolysis with 
hydrochloric acid. The next column gives the ratio of the latter to the former. In 



DETERMINATION OF FOOD VALUE OF PROTEINS. 



'79 



column 5 are given figures for amino nitrogen calculated from the data in column 2, 
while the values in the last column were obtained b)- calculating the per cent calculated 
amino nitrogen of the total soluble nitrogen. 

Table II. — Analysis by SOrensen's Method of Solutions of Cynoscion Rbgalis 

Hydrolyzed by Trypsin. 



Time in 
hours. 


Quantity 

N/io NaOH 

required - 


Quantity 
N/.oNaOH 
required 
after hy- 
drolysis. 


Average 
size of 
peptids. 


Amino 

nitrogen 

calculated. 


.Amino 
nitrogen 

looX 

soluble 
nitrogen. 



H 

I 
2 
s 

8 


c. c. 
34.85 
182.8 
213.8 
232.9 
293-5 
314.8 


c. c. 
125.0 
536.8 
598.0 


5.02 
2.94 
2.80 


0.03S 
•257 
.300 
.327 
.412 
.442 


20- 52 
23-02 
25-87 
27-87 

30- 28 

30.87 


776.0 
787.1 


2.64 
2.50 



From table i it is seen from the ratio of the soluble to the total nitrogen that the 
fish meat goes very rapidly into solution, 74.32 per cent of the nitrogen being in solution 
at the end of a half hour's digestion. Solution, however, is not complete in eight 
hours' time, a fact which is apparently not in harmony with the results of White and 
Crozier," who found with the proteins they studied that all the nitrogen was in the soluble 
form in four to eight hours. The trypsin used, a commercial sample, was of the same 
activity in both series of experiments. These latter exjjeriments were carried on in a 
medium made alkaline with sodium carbonate, while the experiments described in this 
article required sodium hydroxide for reasons already stated. Schierbeck* has proved 
that the action of trypsin in digesting proteins is accelerated by the presence of carbon 
dioxide in solutions which are slightly alkaline, and it is very probably at least 
partly due to this fact that the above differences are found. The variation of the 
proportion of soluble to total nitrogen with increase of time is shown graphically in 
figure I. Extrapolation of the curv^e would indicate that the squeteague would be com- 
pletely dissolved in about 1 4 hours. 

White and Crozier have shown that their artificial digestion experiments gave results 
agreeing closely with those obtained by metabolism work with dogs, rates of digestion 
of different proteins being in the same ratio to each other. Van Slyke and White,'' in a 
study of the relation between the digestion and the retention of ingested proteins, found 
that squeteague is digested more slowly than either beef or cod. From the above facts 
it is fair to conclude that the tardy solution of the squeteague by trypsin shown by our 
data is not alone due to the absence of carbon dioxide, but is a consequence of the 
inherent nature of the protein itself. 

« White & Crozier. op. dt. 

ft Schierbeck. N. P-l Ueber den einfiuss der kohlensaure auf die diastatischen und peptonbildenden fermente im thisrischen 
organismus. Skandinavisches Archivfur Physiologic, bd. 3. s. 344-375. 1892. Leipzig. 

« Van Slyke, D. D. & ^Tiite. G. F-; The relation between the digestibility and the retention of ingested proteins. Journal 
of Biological Chemistry', vol. ix. p. 219-229. 1911. Baltimore. 



i8o 



BULLETIN OF THE bItREAU OF FISHERIES. 



The amino nitrogen in solution increases of course with length of time. The aver- 
age size of the peptids split off from the protein should be especially noted. From 
these data and the experiments of White and Crozier it is evident that the proteins 
studied break down into simple cleavage products practically as soon as they go into 
solution. At the end of a half hour's digestion the average size of the peptids is only 
2.02; the cleavage products on the whole are indicated to be amino acids. The cleav- 
age of certain proteins by trypsin has been intimately studied, and it is known that 



yrog^e/7 






90 



some amino acids 
are readily formed, 
'while others are pro- 
duced slowly or not 
at all. The above re- 
sults, however, show 
that the greater por- 
tion of the nitrogen 
in solution exists in 
bodies of exceed- 
ingly simple char- 
acter. The signifi- 
cance of this physio- 
logically can not be 
pointed out here.but 
will be reserved for 
future discussion. 

The relation of 
the amino to the to- 
tal soluble nitrogen 
is shown in figure 2 . 
After eight hours' 
digestion only 28.35 
percent of the nitro- 
gen is in the amino 
form. This is a con- 
firmation of earlier 

work which, as just mentioned, has shown that there are certain substances which resist 
the hydrolytic action of trypsin altogether. The increase in the proportion of amino 
nitrogen during the eight hours' digestion is very slight, and we must conclude, there- 
fore, that the cleavage of the meat, while yielding amino bodies of a simple nature, 
leaves the greater part of the soluble nitrogen combined in substances which are 
extremely stable. 

The same conclusions may be drawn from the results of our experiments involving 
vSorensen's method. In figure 2 the curves are of the same slope, although, of course, 



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Fig. I. — Change of the per cent of soluble nitrogen of the total nitrogen during the time of 

proteolysis. 



DETERMINATION OF FOOD VALUE OF PROTEINS. 



i8i 



onlv approximate figures are expected on assuming the presence of one amino group 
for every carboxyl group indicated by the sodium hydroxide required. The amino 
nitrogen thus estimated is regularly greater than that determined by the nitrous acid 
method, and the peptids as computed are about 1.5 times as large. It is possible that 
in such calculations • •/ * 

thiseffectis produced j^q^ S^y/iz/A^^/? 



by the presence of 
such monamino- 
dicarboxylic c o m - 
pounds as glutamic 
acid. The discrep- 
ancy in the results 
is not of such a mag- 
nitude as to prevent 
deducing rigid con- 
clusions concerning 
the rate and course 
of digestion of such 
proteins as the one 
under investigation. 

SUMMARY. 



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/}ours 



I. Sorensen's 
method for the de- 
termination of amino 
acids was applied to 
a study of the tryptic 
proteolysis of Cynos- 
cion regalis. The re- 
sults were regular 
and in accord with 
those obtained by the 
nitrous acid method 
for the analysis for 

amino nitrogen. A practical method for the determination of the food value of proteins 
has therefore been developed. 

2. The relatively low rate at which the protein is made soluble agrees with the 
results of metabolism experiments. 

3. Very low cleavage products are formed as soon as the protein goes into solution, 
the average size of the peptids being 2.02 after a half hour's digestion. 

4. There is a very stable nitrogen complex which is not attacked by trypsin. 



/ Z 4 6 S 

Fig. a. — Change of the per cent of amino nitrogen of the soluble nitrogen during the time of 

proteolysis. 



